The double helical structure of DNA requires the existence of a mechanism for unwinding the helix to expose single strands for use as templates by DNA polymerase. One mechanism provided is a class of enzymes, the helicases, that unwind DNA using chemical energy provided by NTP hydrolysis. Four helicases have been described in E. coli, helicases I, II, III and rep protein; the exact role of each in DNA metabolism has not been determined. The long-range goal of this research program is to understand in enzymatic and molecular terms the mechanism of action of each helicase, and to define their role in DNA replication, repair, recombination and conjugation. Helicases are likely involved in all aspects of DNA metabolism. Insight into the role of a specific helicase is gained by determining its DNA substrate requirements; DNA structure at a replication fork is different from the DNA structure present during excision repair. In addition, different helicases may interact with different DNA polymerases. A helicase opening the helix at a replication fork will interact with DNA polymerase III; a helicase unwinding the helix during a repair process may interact with a different DNA polymerase. Interactions between helicase and DNA polymerase increase the rate and processivity of polymerization in other systems and similar effects are expected in E. coli. Study of protein-DNA and protein-protein interactions will also answer questions regarding how the helicases unwind DNA. Specifically, is the unwinding reaction catalytic in mechanism and processive? Must the helicase bind both strands of the duplex to cause unwinding? How is ssb involved in the unwinding reaction? Mutants exist for helicases I, II and rep protein; studies using these mutants have failed to define a role for each protein. Mutants in helicase III are necessary to establish its role and the role of the other helicases in DNA metabolism. Knowledge of the DNA substrate requirements for each protein, the nature of protein-protein interactions and study of the relationship between helicases are required to understand the role of each helicase in DNA metabolism.